The present invention relates to the rapid quantification of bacteria, in particular, of coliform bacteria.
Nearly all water sources contain a wide variety of bacterial contaminants, the great majority of which are not harmful to man. However, mammals frequently add pathogenic organisms to water sources via intestinal excrement. Epidemiological studies have established beyond a doubt that there is a strong correlation between the presence of waterborne pathogenic organisms and the presence of micro-organisms of intestinal origin. Thus, if pathogens are present in a water source, it is almost certain that non-pathogenic intestinal micro-organisms will also be present, generally in much greater concentrations. (On the other hand, the presence of non-pathogenic intestinal organisms does not necessarily imply the presence of pathogenic organisms.)
Of the intestinal organisms present in man, the largest concentration are those bacteria classified as members of the family Enterobacteriaceae. This family consists of five tribes: (1) Eschericheae; (2) Eriwineae; (3) Serrateae; (4) Proteae; and (5) Salmonelleae. Of these tribes, the Eschericheae are the most abundant. Eschericheae are further categorized into three genera: (1) Escherichia; (2) Aerobacter; and (3) Klebsiella. Genera are further classified into species, e.g., Escherichia coli, Escherichia freundii, Enterobacter aerogenes, Aerobacter cloacae, etc.
In order to avoid the problem of analyzing for each of the various species of organism present in water, sanitarians have elected to test for those members of the Enterobacteriaceae family that are gram negative and lactose fermenting. These bacteria are classified as coliform bacteria, without attempting further classification. Of the species meeting these criteria members of the genus Escherichia are the most common, with Escherichia coli predominant.
There is probably no single bacteriological test more frequently performed than that of the examination of water. Yet, with all the vast amount of testing being performed daily, the evaluation still takes anywhere from three to forty eight hours per analysis. A more rapid methodology for bacterial contamination testing appears to be a desirable goal for any application wherein water sanitation may be a problem.
The term "rapid" appears to have varying meanings to microbiologists. The literature lists many papers claiming rapid analysis techniques, where rapid is defined as less than twenty four hours. The present invention relates to a system in which analysis requires less than two hours, preferably less than one hour.
In addition to analysis speed, a water analysis methodology must be specific to coliforms and sensitive enough to detect a bacterial contamination level of 200 bacteria per 100 ml of water or less. Presently available methodologies will not meet the specificity, sensitivity and speed requirements mentioned above.
The standard procedure for bacterial examination of water is given in the American Public Health Association (APHA) publication "Standard Methods for the Examination of Water and Waste Water." The recommended colifirm bacteria detection test procedure consists of a series of three tests: a presumptive test, a confirmed test and a completed test. The presumptive test is a screening test that depends upon the ability of coliform bacteria to oxidize lactose with the production of CO.sub.2 and H.sub.2. The confirmed test attempts to eliminate the bacteria that give false-positive results in the presumptive test. Suspicious results of the confirmed tests are further examined in the completed test to confirm or negate the presence of coliforms. All tests require incubation of a sample to produce an adequate quantity of bacteria for visual analysis of the end result.
There are a number of variations of the APHA test procedure commercially available. However, these also involve long incubation periods.
In addition to the APHA procedure for the detection of coliforms, there are a number of other bacterial detection procedures that have been noted in the literature. Among these are:
a. Radiometric methodologies; PA0 b. Electrochemical methodologies; PA0 c. Chromatographic methodologies; PA0 d. Chemiluminescence methodologies; and PA0 e. Fluorescence methodologies.
Radiometric techniques for coliform detection generally follow the APHA culturing technique with the exception that radioactive C.sup.14 labelled lactose is used. It has been reported that one bacterium can be detected in eight hours.
Electrochemical techniques are somewhat more varied in their detection methodology. Sensitivity of the electrochemical methods are such that reaction times &gt;3 hours and bacterial densities of &gt;10.sup.5 per ml are required for detection.
Chromatographic methodologies are based upon the chemical differences of various types of bacteria. In general, the chromatographic methodologies require &gt;4 hours of culturing to achieve a large enough sample of bacteria for analysis. Also, the technique is probably too difficult to be handled, except by skilled laboratory personnel.
Chemiluminescent methodologies are based upon the presence within bacteria of certain chemicals that either catalyze a light emitting reaction or participate in the reaction. The luminol peroxide reaction is an example of the former, catalyzed by bacterial porphyrins. The luciferin-bacterial ATP (adenosine triphosphate) reaction is an example of the latter. Both reactions are rapid but non-specific. Further, bacterial concentrations &gt;10.sup.3 /ml are required.
There are several flourescent methodologies that have been reported. The use of fluorescent antibodies represents a prime example of a sensitive, specific bacterial analysis test method. Unfortunately, though sensitive, the flourescent antibody technique is much too specific, i.e., it will detect single strains of bacteria but not a mixture of many strains.
A second approach to the use of fluorescence for bacteria detection uses the reaction between fluorescent substrates for bacterial enzymes. In this case, a fluorescent molecule is chemically attached to a bacterial enzyme substrate. In its attached state it is non-fluorescent. However, once reacted upon by the enzyme, the fluorescent molecule is released and can be caused to fluoresce.
A large variety of enzyme-substrate reactions are analyzed by this approach. The reaction between the coliform enzyme .beta.-D-galactosidase and fluorescein-di-(.beta.-D-galactopyranoside) is one example.
Boris Rotman has developed a technique for detecting a coliform enzyme, .beta.-D-galactosidase, at the single molecule level. This enzyme is the key enzyme permitting coliform bacteria to react with galactosides such as lactose, i.e., lactose .sup..beta.-D-galactosidase galactose +glucose. Since the ability to ferment lactose is the primary test for classifying bacteria as coliforms or non-coliforms, the presence or absence of .beta.-D-galactosidase in a bacterium provides a convenient method of coliform detection.
Rotman's technique involves the use of a fluorescent reagent that acts as a substrate for .beta.-D-galactosidase. The reagent is fluorescein-di-(.beta.-D-galactopyranoside). When this reagent is hydrolyzed by bacterial .beta.-D-galactosidase, the fluorescein portion of the molecule is released. Fluorescein, being a highly fluorescent material, is readily detected at the part per billion level.
Fluorescein, produced by the hydrolysis reaction, rapidly diffuses out of a coliform bacterium. Even at the part per billion detection level, if the fluorescein is allowed to diffuse into a volume of water, orders of magnitude larger than a bacterium, the concentration of fluorescein will be orders of magnitude lower than the detection limit. Rotman solved this dilution problem by containing small numbers (1-5) of E. coli within microdroplets of water. The microdroplets are in turn surrounded by silicone oil in which the fluorescein is insoluble. Thus, all of the fluorescein produced is contained within a very small volume, typically 10.sup.-12 liters per droplet.
The minimum number of molecules of fluorescein that can be detected, based upon a convervative 10.sup.-5 moles per liter detection limit and a 10.sup.-12 liter droplet size, is estimated at 6.023.times.10.sup.6 molecules using the relationship: EQU n=A .multidot..alpha..multidot.v (1)
where
n=minimum number of molecules required for detection
A=Avogadro's number--6.023.times.10.sup.23 molecules per mole
.alpha.=detection limit--moles per liter
v=volume of sample--liters
The time required to form 6.023.times.10.sup.6 molecules of fluorescein can be estimated at about 100 seconds from the turnover rate of the .beta.-D-galactosidase molecule (i.e., the number of molecules of lactose hydrolyzed per second) and a knowledge of the average number of enzyme molecules per bacterium. EQU t =n/.phi..multidot..rho. (2)
where
t=time required
.phi.=turnover of .beta.-D-galactosidase--120 molecules per second
.rho.=number of molecules of enzyme per bacterium--500
Thus, by the use of a highly specific reagent and a simple technique for concentrating the fluorescent product of that reagent's reaction, it is possible to detect single coliform bacteria in reaction times of less than 5 minutes. In fact, the technique is so sensitive that single molecules of enzyme, entrapped within microdroplets, can be detected in less than 10 hours of reaction time.
While the Rotman technique is sensitive, it is time consuming and requires skilled personnel. It is an object of the present invention to provide a method and apparatus for quantification of bacteria which can be performed rapidly and by unskilled personnel. By "rapidly" is meant less than two hours at a bacteria level of 200 per 100 ml of water or less. The foregoing and other objects which will be apparent to those having ordinary skill in the art are achieved by the present invention, a description of which follows.